JOURNAL OF THE

WORLD AQUACULTURE SOCIETY

Vol. 38, No. 2

June, 2007

Kinetics of Cellular Component in Inflammatory Response Induced

by Different Stimuli in the Swim Bladder of Pacu, Piaractus
mesopotamicus Holmberg 1887 (Characidae)
F. R. BOZZO
Department of Veterinary Pathology, Sao Paulo State University (Unesp),
Jaboticabal, Sao Paulo, Brazil

J. R. E. MORAES

AND

FLAVIO RUAS

DE

MORAES1

Department of Veterinary Pathology, Sao Paulo State University (Unesp),

Jaboticabal, Sao Paulo, Brazil,
and Aquaculture Centre, Sao Paulo State University (Unesp), Jaboticabal, Sao Paulo, Brazil

G. T. PEREIRA
Department of Biostatistic, Sao Paulo State University (Unesp), Jaboticabal, Sao Paulo, Brazil

M. TAVARES-DIAS

AND

E. M. ONAKA

Aquaculture Centre, Sao Paulo State University (Unesp), Jaboticabal, Sao Paulo, Brazil
Abstract.The present study assessed the kinetics of
cell accumulation at the site of inflammation induced by
thioglycolate, Escherichia coli lipopolysaccharide (LPS)
and heat-inactivated Aeromonas hydrophila, in the pacu,
Piaractus mesopotamicus (Characidae), swim bladder. A
quantitative, as well as qualitative, assessment was done of
all the cells present in the exudate at 6, 24, and 48 h
(n 5 8) after inoculation of inflammatory agents. The
results show that the thioglycolate was the irritant to induce
higher total inflammatory cell accumulation when compared to the control group, 6 h after insult (P , 0.05).
Inoculation of heat-inactivated Aeromonas hydrophila
induced progressive accumulation of total inflammatory
cells, with cell number peaking after 24 h and being
significantly higher than observed in the other groups
(P , 0.05). Injection of LPS also induced greater cell
accumulation when compared to the control group
(P , 0.05), although in lower numbers than those induced
by the other two irritants. All irritants injected induced
significantly greater accumulation of lymphocytes and
thrombocytes when compared to the control group
(P , 0.05).

The first inflammatory phenomenon described in fish was the phagocytosis of guinea
pig erythrocytes and of Bacillus anthracis by
peritoneal mononuclear cells (Metchnikoff
1893, 1905; Mesnil 1895). Afterward, many authors tried to characterize the cells found present
at inflammation sites induced by various irritat1

Corresponding author.

ing substances in several fish species at different

observational periods. The intraperitoneal injection of kerosene in rainbow trout, Oncorhynchus mykiss, induced neutrophil accumulation
(Weinreb 1958) and Yersinia ruckeri provoked
accumulation of 57% of lymphocytes and 43%
of polymorphonuclears (Griffin 1983). Infiltration of lymphocytes and macrophages was
observed in gold fish, Carassius auratus, after
intramuscular injection of silica (2%) (Jansson
and Waaler 1967). Neutrophils and macrophages accumulated after injections of oyster
glycogen or Vibrio alginolyticus in plaice, Pleuronectes platessa (MacArthur et al. 1984), while
the injection of paraffin in the peritoneal cavity
of Nile tilapia, Oreochromis niloticus, induced
the infiltration of neutrophils only (Suzuki
1986). The acute inflammation induced in
Atlantic salmon, Salmo salar, by glucane, oyster
glycogen, or incomplete Freunds adjuvant in
the peritoneal cavity resulted in high counts of
macrophages, neutrophils, and, for the first time,
thrombocytes (Jorgensen et al. 1993). The injection
of carrageenin into the swim bladder of Nile
tilapia, and pacu, Piaractus mesopotamicus,
induced the accumulation predominantly of
thrombocytes, and of a lesser amount of macrophages and a small number of granulocytes
(Matushima and Mariano 1996; Martins 2000).

Copyright by the World Aquaculture Society 2007

302

KINETICS OF CELLULAR COMPONENT IN THE SWIM BLADDER OF PACU

These facts show how complex the characterization of cells that participate in the inflammatory
process in fish can be. Different observations
or results can be because of many variables,
starting by fish species considered and its
phylogenetic position, as well as of difficulties
in distinguishing different cell types as observed
by Jorgensen et al. (1993) and Matushima
and Mariano (1996), for which the use of cytochemical methods is indispensable.
The descriptions of the previous authors
regarding the cellular types are confusing and
the different types of noxious stimulants require
clarification. Based on the former considerations, the present study aimed to attain a
quantitative, morphological, and cytochemical characterization of the cell population in
inflammation induced by thioglycolate and
Escherichia coli lipopolysaccharide (LPS) and
inactivated Aeromonas hydrophila used as irritants, injected into the pacu, swim bladder.
The species chosen was a native teleost fish of
the Parana-Paraguay Basin, of importance in
Brazil in relation to human consumption,
angling, and aquaculture.
Materials and Methods
Fish used for this study were 96 young pacu
all from the same hatching group, with a mean
individual weight of 115.0 6 1.0 g and of
17 6 1.21-cm total length, maintained in 250-L
tanks with nonchlorinated running water. Water
temperature in the tanks ranged from 28.1 to
31.0 C, pH from 6.5 to 7.0, dissolved oxygen
at 3.55.0 mg/L.
The fish were distributed into four groups
each group containing 25 animals: G1 injected
with 0.5 mL of a 0.65% sterile sodium chloride
solution (control), G2 injected with 0.5 mL of
a 6% thioglycolate saline solution, G3 injected
with 3 3 109 colony-forming units of heatinactivated A. hydrophila (30-min bath at 40 C)
dissolved in 0.5 mL of a 0.65% saline solution,
and G4 injected with 3.0 mg/kg of E. coli
LPS dissolved in 0.5 mL of a 0.65% saline
solution.
The fish were sedated by immersion in
a 1.0 g/L benzocaine aqueous solution (Noga
1996) and the irritants were injected into the

303

anteromedial region with a sterile needle and

syringe, accessing the anterior swim bladder.
Following these procedures the fish were immersed in a 10 g/L sodium chloride solution for
15 min, and, for the next couple of days, new
baths for 15 min in a 1.0 g/L solution stimulating the mucus production of skin (Petric et al.
2003).
Total and differential counts of all cells present in the inflammatory exudates were done 6,
24 and 48 h after application of irritating stimuli.
The fish were euthanatized by deepening of
anesthesia and then dissected. Swim bladders
were collected and internally washed with an
injection of 0.5 mL of phosphate-buffered
saline solution containing 0.01 mL of 5% ethylenediaminetetraacetic acid. The same volume of
solution was collected using a Pasteur pipette
and then transferred to centrifuge tubes and kept
on ice. One aliquot of this volume was then
transferred to a Newbauer chamber for total cell
count. For the differential cell counting of
thrombocytes, lymphocytes, macrophages, and
granulocytes, the exudate was centrifuged at
1000 rpm for 5 min in a clinical centrifuge.
The supernatant was then discarded and total
sediment was collected with a Pasteur pipette
and deposited on a glass slide for postfixation
by methyl alcohol. The slides were stained using
a MayGrunwaldGiemsaWright (MGGW)
stain (Tavares-Dias and Moraes 2003) for posterior cell count by light microscopy. Up to 100
cells were counted, including all the different
cell types found aggregated at the inflammatory
site. Other smears were stained by the PAS
method for visualization of carbohydrates
(McManus 1946; Lison 1960).
Statistical analysis was done entirely by random projection. Comparison of averages was
done by Tukeys test, with 5% of probability.
Results
On macroscopic observation, fish that received thioglycolate or A. hydrophila in this study
all showed accumulation of exudate in their
swim bladders. This was of yellow coloring
and gelatinous consistency. The fish that
received LPS and the controls did not show
these alterations.

304

BOZZO ET AL.

The number of cells collected in the inflammatory exudate exceeded those in the control
group (P , 0.05) 6 h after insult for all different irritants. The total cell count in fish that
received thioglycolate and A. hydrophila was
higher than that in the control group (P , 0.05)
found 24 h after insult. In the fish injected with
LPS, infiltrated cells were fewer in smaller number (P , 0.05) when compared to those receiving inoculations of A. hydrophila but did not
differ statistically from the control group. The
inoculation of A. hydrophila caused a higher
aggregation of cells (P , 0.05) 48 h after insult
as observed in the control group and in the LPSinjected fish.
The total number of inflammatory cells gradually decreased in fish from the same treatments, 648 h after insult, except in the fish
inoculated with A. hydrophila (Table 1).
The injection of thioglycolate in the swim
bladder caused accumulation of more thrombocytes (P , 0.05) after 6 h than that observed
in the control group (Table 2). After 24 h, all
the irritants induced accumulation of a higher
number of thrombocytes (P , 0.05) as in the
control group.
All fish injected with thioglycolate and A. hydrophila presented higher numbers of thrombocytes (P , 0.05) at the inflammatory site after
48 h in comparison to the control group. A
decrease in thrombocyte count (P , 0.05) was
verified 24 h after insult in the fish receiving
thioglycolate. The same occurred in the fish
injected with LPS, although after 48 h (Table 2).
The injections of thioglycolate and A. hydrophila induced a larger infiltration of granulocytes (P , 0.05) after 24 h in comparison to

the control group. The only difference observed

between the different treatments during the
studied time period was in the fish injected with
thioglycolate, where the number of granulocytes
found was lower (P , 0.05) after 48 h
(Table 2).
Injection of thioglycolate and A. hydrophila
in fish caused greater accumulation (P , 0.05)
of lymphocytes when compared to the controls
after 6 and 24 h. Independently of the irritant
used, however, no significant difference (P .
0.05) was observed in the lymphocyte count
after 48 h when comparing treatment groups.
A gradual decrease in lymphocyte number was
observed for all treatments during the observational period, up to 48 h. This decrease was significant (P , 0.05), however, exclusively in the
group of fish injected with LPS (Table 2).
Fish injected with A. hydrophila showed
higher number of macrophages in exudate (P ,
0.05) than that observed in the control group
only after 24 h. During the whole observational
period, no statistically significant difference
was seen at any of the other observation times
(Table 2). In the exudate extensions, independent of which irritant was inoculated,
lymphocytes, macrophages, granulocytes, and
thrombocytes were identified. The lymphocytes
were seen as small cells with a high nucleus/
cytoplasm ratio when stained by MGGW (Fig. 1
A). Macrophages were the largest cells observed
in the exudates and exhibited cellular pleomorphism when stained by MGGW (Fig. 1B).
Granulocytes stained by MGGW showed
elliptical nuclei with small nonstained cytoplasmic granules (Fig. 1C). Thrombocytes were
identified as medium sized when compared to

TABLE 1. Values of absolute averages (mL) 6 SE of mean total count of cells present in the swim bladder of pacu,
Piaractus mesopotamicus, injected with thioglycolate (Tio), Escherichia coli lipopolysaccharide (LPS), or Aeromonas
hydrophila (bacteria). Jaboticabal, Brazil, 2004.1
Observation time (h)
Treatments

Total cells

Control
Tio
LPS
Bacteria

6
458
3758
1525
2863

339Ca
339ABa
339Ba
339Aa

24
120
2200
900
3553

387Cab
339ABab
339BCab
339Aa

48
242
717
208
1833

339Bb
449ABb
339Bb
339Aa

1 Values followed by the same letters do not differ by Tukeys exact test (P , 0.05). Uppercase letters for comparison of
treatments and lowercase letters for comparison of time periods, in the same treatment.

305

KINETICS OF CELLULAR COMPONENT IN THE SWIM BLADDER OF PACU

TABLE 2. Absolute averages (mL) 6 SE of counts of inflammatory cells in the swim bladder of pacu, inoculated with
thioglycolate (Tio), Escherichia coli lipopolysaccharide (LPS) or Aeromonas hydrophila (bacteria). Values followed by
the same letters do not differ by Tukeys exact test (P , 0.05). Jaboticabal, Brazil, 2004.1
Observation time
Variables

Granulocytes

Lymphocytes

Thrombocytes

Macrophages

Treatments
Control
Tio
LPS
Bacteria
Control
Tio
LPS
Bacteria
Control
Tio
LPS
Bacteria
Control
Tio
LPS
Bacteria

6
148
387
159
204
84
1109
421
1409
153
2121
851
853
21
60
36
58

89Aa
88Aab
89Aa
89Aa
129Ba
12Aa
129ABa
12Aa
173Ba
173Aa
173ABa
173ABa
58Aa
58Aa
58Aa
58Aa

24
64
1253
159
445
1
557
203
1014
31
254
488
1141
2
106
19
955

101Ca
89Aa
89BCa
101ABa
48Bb
129Aa
129Aa
148Aa
198Bb
173Ab
173Aa
198Aa
66Ba
58Aab
66Ba
58Aa

48
84
214
59
234
98
74
38
544
32
363
74
967
22
62
9
89

89Aa
117Ab
89Aa
89Aa
129AB
171ABa
171Bb
171Aa
173Cab
229ABab
229Bbc
229Aa
58Aa
77Aa
77Aa
77Aa

1 Uppercase letters for comparison of treatments and lowercase letters for comparison of time periods, in the same
treatment.

the macrophage cells. They presented a small

nucleus:cytoplasm ratio and nuclei appearing
mainly at the cells perimeter. The cytoplasm
showed itself to be acidophilic when stained
by MGGW (Fig. 1D).
Discussion
The fish that received thioglycolate or A. hydrophila in this study all showed accumulation
of exudate in their swim bladders. This was of
yellow coloring and gelatinous consistency, a
fact not observed in the fish injected with LPS
nor in the noninjected controls. These results
demonstrate a more intense inflammation after
insult, in groups injected with thioglycolate
and A. hydrophila. The findings are in agreement with the reports of the effect of carrageenin in pacu, swim bladders (Martins 2000).
The number of total cells observed in the
pacu, swim bladders, was four to seven times
increased after 24 h in the fish injected with
thioglycolate or A. hydrophila, respectively. A
similar fact was observed in the peritoneal cavity of plaice, where the number of total leucocytes was seen to be three times the initial
value after inoculation of oyster glycogen and

V. alginolyticus (MacArthur et al. 1984). In

Atlantic salmon injected with glucane, a 20
30 times increase in leukocyte number was
observed (Jorgensen et al. 1993). In tambacu
hybrids, P. mesopotamicus 3 Colossoma macropomum, the injection of carrageenin duplicated the leukocyte count in swim bladder
exudate (Martins et al. 2001). These reports
show that accumulation of cells at the inflammatory site in fish is a relevant fact, although the
numbers are incomparably smaller than those
observed in the inflammatory process in laboratory rats and mice (Moraes and Garcia-Leme
1982; Moraes et al. 1987, 1993).
The presence of macrophages in inflammatory exudate of fish is low or inexistent
(MacArthur et al. 1984; Suzuki 1986; Afonso
et al. 1997). On the other hand, the intraperitoneal injection of glucane, oyster glycogen, or
incomplete Freunds adjuvant in Atlantic
salmon resulted in a high thrombocyte count
in the peritoneal cavity (Jorgensen et al. 1993).
Thrombocytes were also present in inflammatory exudate of sea bass, Dicentrarchus labrax
(Vale et al. 2002). In Nile tilapia, inoculated
with carrageenin, cellular exudate compromised

306

BOZZO ET AL.

FIGURE 1. Photomicrograph of cells found in exudate 6 h after injection of irritants. A) Lymphocyte after lipopolysaccharide (LPS); B) macrophage after LPS; C) granular leukocyte after thioglycolate; D) thrombocyte after saline
solution. Stain: MayGrunwaldGiemsaWright. Bar: 12.5 mm.

63.0% thrombocytes and 17.0% macrophages,

3 h after insult (Matushima and Mariano 1996).
The same experimental protocol in pacu showed
the presence of 70.5% thrombocytes and 31.0%
macrophages in inflammatory exudate, 6 h after
an injection of carrageenin, these numbers not
being at all expressive (Martins 2000).
In the present study with pacu, a marked
presence of thrombocytes was observed as well
as a smaller number of lymphocytes, while
the macrophages were seen in small numbers
already at initial assessment. Former studies are
controversial in this aspect because of variables
related to the considered fish species, the methods and irritants used for inducing inflammation,
and methods for collecting, preserving, identifying, and counting the cells found in the inflammatory exudates. All cell types described, however,

showed interesting functions when considering

the hosts defense mechanism. Furthermore, the
fact that in fish, the inflammatory response is
not of the same magnitude nor is patterned as occurs in mammals should be held in consideration.
In macrophages from inflammatory exudate
from pacu, in this study, glycogen granules were
not identified after application of the PAS
method as was reported in the rainbow trout
(Afonso et al. 2000). However, in exudate from
sea bass induced with Photobacterium damselae, macrophages and neutrophils showed
evidence of glycogen granules, making the distinction of these cell populations by light
microscopy difficult. Both cells could be distinguished only by peroxidase patterns, which
occur in neutrophils but not in macrophages
(Vale et al. 2002).

KINETICS OF CELLULAR COMPONENT IN THE SWIM BLADDER OF PACU

In inflammatory exudate from Nile tilapia

(Matushima and Mariano 1996) and pacu,
injected with carrageenin, some leukocytes were
described only as granulocytes (Martins 2000).
Other studies clearly show the phagocytic role
of thrombocytes in fish (Suzuki 1986; Jorgensen
et al. 1993; Hill and Rowley 1996). Furthermore, the role of thrombocytes in the organisms
defense mechanism is reinforced by their adherence to A. hydrophila (Kozinska et al. 1999).
Fish thrombocytes interact with nonimmune
and immune cells such erythrocytes potentiating
the formation of rosettes around a central macrophage, suggesting that thrombocytes may represent a link between innate and adaptive
immunity (Passantino et al. 2005). Thus, based
on this study and others in related bibliography,
the presence of thrombocytes at the inflammatory site probably is not random, as could be initially thought. The phenomenon repeats itself,
and many authors, although using different
methodologies, showed their presence in inflammatory exudate from many fish species. These
cells are evident because of their phagocytic activity at the inflammatory site and in circulating
blood, and by presenting enzymes that are important to the phagocytic process. Thus, this evidence strongly suggests that thrombocytes,
along with leukocytes, besides presenting hemostatic functions, also act as defense cells in fish.
Acknowledgments
The authors thank the National Council of
Technological and Scientific Development
(CNPq) for financial support, and the administrative directors and technicians of the Caunesp
(Aquaculture Centre of Unesp) for use of the
aquarium facilities. Thanks are due to Maria
Ines Y. de Campos and Francisca de Assis
Ardison for excellent technical assistance.
Literature Cited
Afonso, A., A. E. Ellis, and M. T. Silva. 1997. The
leucocyte population of the unstimulated peritoneal
cavity of rainbow trout (Oncorhynchus mykiss). Fish
and Shellfish Immunology Aberdeen 7:335348.
Afonso, A., P. M. Macedo, A. E. Ellis, and M. T. Silva.
2000. Glycogen granules in resting and inflammatory

307

rainbow trout phagocytes an ultrastructural study.

Diseases of Aquatic Organisms 42:101110.
Griffin, B. R. 1983. Opsonic effect of rainbow trout (Salmo
gairdneri) antibody on phagocytosis of Yersinia ruckeri by trout leukocytes. Developmental and Comparative Immunology 7:253259.
Hill, D. J. and A. F. Rowley. 1996. The thromboxane
mimmetic, U-46619, induces the aggregation of fish
thrombocytes. Brazilian Journal Haematol Oxford
92:200211.
Jansson, C. W. Jr. and E. Waaler. 1967. Body temperature, antibody formation and inflammatory response.
Acta Pathologica et Microbiologica Scandinavica
69:577566.
Jorgensen, J. B., H. Lunde, and B. Robertsen. 1993.
Peritoneal and head kidney cell response to intraperitoneally injected yeast glucan in Atlantic salmon,
Salmo salar, L. Journal of Fish Diseases 16:313325.
Kozinska, A., J. Antychowicz, and P. Kostrzewa. 1999.
Influence of thrombocyte activity on the susceptibility
of the carp (Cyprinus carpio L.) to Aeromonas hydrophila infection in different temperature. Bulletin of
Veterinary Institute in Pulawy 43:4353.
Lison, L. 1960. Lipides et lipoproteines. In L. Lison, editor.
Histochemie et cytochimie animales. Principes et
methods. GauthirVillars, Paris, France.
MacArthur, J. I., T. C. Fletcher, B. J. S. Pirie,
R. J. L. Davison, and A. W. Thomson. 1984.
Peritoneal inflammatory cells in plaice, Pleuronectes
platessa L.: effects of stress and endotoxin. Journal of
Fish Biology 25:6981.
Martins, M. L. 2000. Efeito da suplementac
xao com
vitamina C sobre a reac
xao inflamatoria em Piaractus
mesopotamicus Holmberg, 1887 estressados. Dc Dissertations, University of Sao Paulo State, Aquaculture
Centre of Unesp, Jaboticabal/SP, Brazil.
Martins, M. L., E. M. Onaka, M. Tavares-Dias,
F. R. Bozzo, and E. B. Malheiros. 2001. Caractersticas hematologicas do hbrido de tambacu, seis E 24
horas apos a injec
xao de substancias irritantes na bexiga
natatoria. Revista de Ictiologia 9(1/2):2531.
Matushima, E. R. and M. Mariano. 1996. Kinects of the
inflammatory reaction induced by carrageen in the
swimbladder of Oreochromis niloticus (Nile tilapia).
Brazilian Journal of Veterinary Research Animal
Science 33(1):510.
McManus, J. F. A. 1946. Histological demonstration of
mucin after periodic acid. Nature 158:202.
Mesnil, F. 1985. Sur le mode des resistence des vertebrades
inferieures aux invasions microfiennes. Annals of
Institute Pasteur, 2:301311.
Metchnikoff, E. 1893. Lectures on the comparative
pathology of inflammation. Delivered at the Pasteur
Institute in 1891. English translation by F. A. and
E. H. Starling, Kegan Paul, Trench, Trubner, London,
UK.
Metchnikoff, E. 1905. Immunity in infective diseases.
University Press.

308

BOZZO ET AL.

Moraes, F. R. and J. Garcia-Leme. 1982. Endogenous

corticosteroids and insulin in acute inflammation.
Microvascular Research 23:281283.
Moraes, F. R., G. H., Bechara, and J. R. M. Moraes.
1987. Effect of alloxan diabetes and adrenalectomy on
carrageenin-induced pleurisy in the rat. Brazilian
Journal of Medicine and Biological Research 20(1):
4753.
Moraes, J. R. E., F. R. Moraes, and G. H. Bechara. 1993.
Macrophage participation in the potentiation of
carrageenin-induced peritonitis in rats pre-treated with
chloramphenicol succinate. Brazilian Journal of Medicine
and Biological Research 26:493507.
Noga, E. J. 1996. Fish disease: diagnostic and treatment,
1st edition. Mosby, St. Louis, Missouri, USA.
Passantino, L., A. Cianclotta, R. Patruno, M. R. Ribaud,
E. Jirillo, and G. F. Passantino. 2005. Do fish
thrombocytes play an immunologcal role? Their cytoenzymatic profiles and function during an accidental
piscine candidiasis in aquarium. Immunopharmacology and Immunotoxicology 27:345356.
Petric, M. C., M. L. Martins, E. M. Onaka, J. R. E.
Moraes, F. R. Moraes, and E. B. Malheiros. 2003.

Suplementacxao alimentar com vitamina C potencializa

a formac
xao de macrofagos policariontes em Piaractus
mesopotamicus Holmberg, 1887 (Osteichthyes: Characidae). Boletim do Instituto de Pesca 29(1):6976.
Suzuki, K. 1986. Morphological and phagocytic characteristics of peritoneal exudate cells in tilapia, Oreochromis niloticus (Trewavas), and carp, Cyprinus carpio L.
Journal of Fish Bilogy 29(3):349364.
Tavares-Dias, M. and F. R. Moraes. 2003. Caractersticas
hematologicas da Tilapia rendalli Boulenger, 1896
(Osteichthyes: Cichlidae) capturada em Pesque-Pague
de Franca, Sao Paulo, Brasil. Bioscience Journal
19:103110.
Vale, A., A. Afonso, and M. T. Silva. 2002. The professional phagocytes of sea bass (Dicentrarchus labrax
L.): cytochemical characterisation of neutrophils and
macrophages in the normal and inflamed peritoneal
cavity. Fish Shellfish and Immunology 13:183198.
Weinreb, E. L. 1958. Studies on the histology and histopathology of the rainbow trout, Salmo gairdneri
irideus. I. Hematology: under normal and experimental conditions of inflammation. Zoologica 43:
145154.